A central problem in developmental biology concerns the mechanisms that govern the formation of elaborate organs from seemingly uniform primordial tissues. The vertebrate eye, for instance, develops from the homogeneous optic neuroepithelium that under the influence of extracellular growth factors becomes separated into the future retina, retinal pigment epithelium (RPE), iris, and optic stalk. We found that the optic neuroepithelium initially co-expresses a set of transcription factors which through differential regulation of expression become segregated into distinct anatomical locations that intimate the future parts of the eye. Among these factors is one, MITF, that we found to be present in multiple isoforms generated by alternative promoter/exon use from a single gene. To address the question of the role and regulation of these isoforms, we determined their developmental expression profiles in the developing mouse eye and analyzed the role of some of them in genetic models. The studies indicate that the regulation of MITF in the eye is isoform-selective and that some isoforms are more critical than others in effecting normal retina and RPE development. Moreover, in genetic models we found that other transcription factors that are critical regulators of MITF and in turn are being regulated by MITF help to determine and refine the boundaries between the RPE and the optic stalk and between the RPE and the retina. Hence, the studies have revealed insights into a complicated crosstalk between transcription factors during eye development that will become important in any future attempt to specifically reprogram multipotent cells in the eye for cell-based replacement therapies.